Biochem 717
Gene Cloning
Prof Amer Jamil
Dept of Biochemistry
University of Agriculture
Faisalabad
How to construct a recombinant
DNA molecule?
• DNA isolation
• Cutting of DNA molecule with the help of restriction enzymes
• Transfer of DNA molecule into a suitable vector
With the help of DNA ligases
Transformation of recombinant molecule into suitable host like E. coli
• Production of large number of copies of the recombinant molecule in the host
• Checking the gene expression
By Prof Amer Jamil
Overexpression of proteins
• TYPE I: Restrict away from recognition
site
•TYPE II: Restrict within recognition site
•TYPE III: Restrict away from recognition
site
Restriction endonucleases
• Also called restriction enzymes
• Occur naturally in bacteria
• Hundreds are purified and available commercially
• Named for bacterial genus, species, strain, and type
Example: EcoRI
Genus: Escherichia
Species: coli
Strain: R
Type II Restriction endonucleases
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Restriction Endonuclease Specificity
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY at
that sequence
– They recognize 4-bp, 6-bp,
8-bp palindromic sequences
– The frequency of cuts
lessens as the recognition
sequence is longer
– They cut DNA reproducibly
in the same place
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Restriction-Modification System
• What prevents these enzymes from cutting up the host DNA?– They are paired with
methylases
– Theses enzymes recognize, methylate the same site
• Together they are called a restriction-modification system, R-M system
• Methylation protects DNA, after replication the parental strand is already methylated
• Star activity
• Buffer systems
• Enzyme activity
• Isoschizomers
• dam/dcm sensitivity
Cutting and
Joining DNA
Fragments
Restriction
Enzyme: cleaves
DNA at specific
sequences
DNA Ligase: joins
DNA fragments by
sealing nicks in
backbone
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Terminal Transferase
• cDNAs don’t have the sticky ends of genomic
DNA cleaved with restriction enzymes
• Blunt ends will ligate, but is inefficient
• Generate sticky ends using enzyme terminal
deoxynucleotidyl transferase (TdT), terminal
transferase with one dNTP
– If use dCTP with the enzyme
– dCMPs are added one at a time to 3’ ends of the cDNA
– Same technique adds oligo(dG) ends to vector
– Generate ligation product ready for transformation
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Restriction Mapping
• Prior to the start of large-scale sequencing
preliminary work is done to locate
landmarks
– A map based on physical characteristics is
called a physical map
– If restriction sites are the only map features
then a restriction map has been prepared
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Restriction Map Example
• Consider a 1.6 kb piece of DNA as an example
• Cut separate samples of the original 1.6 kb fragment with different restriction enzymes
• Separate the digests on an agarose gel to determine the size of pieces from each digest
• Can also use same digest to find the orientation of an insert cloned into a vector
About vectors!
• They have selectable markers, origin of replication and increased copy numbers
• They can carry different sizes of DNA molecules
• Cosmids carry large sized DNA molecules
• YAC carry even larger pieces of DNA
Commonly used vectors
• pUC18 (Expression vector)
• pBR322 (cloning vector)
• M13 (sequencing vector)
• Lambda vector
• Agrobacterium tumefaciencs (used for
plants)
• YAC
Cloning Vectors
• For Bacterial Hosts
– Bacteriophage
– Cosmid
– Expression Vector
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Plasmids As Vectors
• pBR plasmids were developed early but
are rarely used today
• pUC series is similar to pBR
– 40% of the DNA has been deleted
– Cloning sites are clustered together into one
area called the multiple cloning site (MCS)
– MCS allows one to cut the vector and foreign
gene with two different restriction enzymes
and use a directional cloning technique to
know the orientation of the insert
Useful Plasmid
Features
•Relaxed Replication
•Selectable Markers
•Streamlined
•Polylinker or MCS
• Identification of
Recombinants
•most derived from
pUC or pBR322
|SacI| |ScII| |XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI| |SalI||XhoI| |KpnI|
GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC
CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG
Multiple Cloning Site:
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Screening: antibiotics and b-galactosidase
Screening capabilities within plasmids:
– Antibiotic resistance genes (i.e., ampicillin resistance
gene) allow for the selection of bacteria that have
received a copy of the vector
– Multiple cloning site inserted into the gene lacZ’ coding
for the enzyme b-galactosidase
• Clones with foreign DNA in the MCS disrupt the ability of the
cells to make b-galactosidase
• Plate on media with a b-galactosidase indicator (X-gal) and
clones with intact b-galactosidase enzyme will produce blue
colonies
• Colorless colonies should contain the plasmid with foreign DNA
compared to blue colonies that do not contain the plasmid with
DNA
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Phages As Vectors
• Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another
• Phage vectors infect cells much more efficiently than plasmids transform cells
• Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area
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l Phage Vectors
• First phage vectors were constructed by Fred
Blattner and colleagues
– Modifications included removal of the middle region and
retention of the genes needed for phage replication
– Could replace removed phage genes with foreign DNA
• Advantage: Phage vectors can receive larger
amounts of foreign DNA (up to 20kb of DNA)
– Traditional plasmid vectors take much less
• Phage vectors require a minimum size foreign
DNA piece (12 kb) inserted to package into a
phage particle
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Two Paths of Phage Reproduction
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Lysogenic Mode
• A 27-kD phage protein (l repressor, CI) appears
and binds to 2 phage operator regions
• CI shuts down transcription of all genes except
for cI, gene for l repressor itself
• When lysogeny is established the phage DNA
integrates into the bacterial genome
• A bacterium harboring integrated phage DNA is
called a lysogen and the integrated DNA is
called a prophage
• The phage DNA in the lysogen replicates along
with the host DNA
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Lytic Reproduction of Phage l
• Lytic reproduction cycle of phage l has 3
phases of transcription:
– Immediate early
– Delayed early
– Late
• Genes of these phases are arranged
sequentially on the phage DNA
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Genetic Map of Phage l
• DNA exists in linear form in the phage
• After infection of host begins the phage DNA circularizes
• This is possible as the linear form has sticky ends
• Gene transcription is controlled by transcriptional switches
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Antitermination and Transcription
One of 2 immediate early
genes is cro
– cro codes for a repressor
of cI gene that allows
lytic cycle to continue
– Other immediate early
gene is N coding for N,
an antiterminator
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Establishing Lysogeny
• Phage establish lysogeny by:
– Causing production of repressor to bind to early operators
– Preventing further early RNA synthesis
• Delayed early gene products are used
– Integration into the host genome
– Products of cII and cIII allow transcription of the cI gene and production of l repressor
• Promoter to establish lysogeny is PRE
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Model of Establishing Lysogeny
• Delayed early transcription from PR produces cII
mRNA translated to CII
• CII allows RNA polymerase to bind to PRE and
transcribe the cI gene, resulting in repression
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Maintaining Lysogeny
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Cloning Using a Phage Vector
hfl- selection
• HflA decreases cII stability….hence lytic
mode
• hflA- strain…..more suppressor….No
plaques
• cI within MCS……
– With insert…… lytic
– Without insert….lysogenic (in hflA- strain)
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Cosmids
Cosmids are designed for cloning large DNA
fragments
– Behave both as plasmid and phage and contain
• cos sites, cohesive ends of phage DNA that allow the
DNA to be packaged into a l phage head
• Plasmid origin of replication permitting replication as
plasmid in bacteria
– Nearly all l genome removed so there is room
for large inserts (40-50 kb)
– Very little phage DNA yields them unable to
replicate, but they are infectious and carry their
recombinant DNA into bacterial cells
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M13 Phage Vectors
• Long, thin, filamentous phage
• Contains:
– Gene fragment with b-galactosidase
– Multiple cloning site like the pUC family
• Advantage
– This phage’s genome is single-stranded DNA
– Fragments cloned into it will be recovered in
single-stranded form
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M13 Cloning to Recover Single-stranded
DNA Product
• After infecting E. coli cells,
single-stranded phage DNA is
converted to double-stranded
replicative form (RF)
• Use the replicative form for
cloning foreign DNA into MCS
• Recombinant DNA infects host
cells resulting in single-stranded
recombinant DNA
• Phage particles, containing
single-stranded phage DNA is
secreted from transformed cells
and can be collected from media
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Phagemids
Phagemids are also vectors
– Like cosmids have aspects of
both phages and plasmids
– Has MCS inserted into lacZ’
gene to screen blue/ white
colonies
– Has origin of replication of
single-stranded phage f1 to
permit recovery of single-
stranded recombinant DNA
– MCS has 2 phage RNA
polymerase promoters, 1 on
each side of MCS
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Eukaryotic Vectors and Very High
Capacity Vectors
• There are vectors designed for cloning
genes into eukaryotic cells
• Other vectors are based on the Ti plasmid
to carry genes into plant cells
• Yeast artificial chromosomes (YAC) and
bacterial artificial chromosomes (BAC) are
used for cloning huge pieces of DNA
Ti plasmid and YAC
Vector Insert (kb) Host Copy No.
P1 70-100 E. coli 1 Bacteriophage P1
PAC 130-150 E. coli 1 P1 artificial
chromosome
BAC 120-300 E. coli 1
YAC 250-400 Yeast 1